Betulinic acid derivatives that target gp120 and inhibit multiple genetic subtypes of human immunodeficiency virus type 1

Betulinic acid (BA) derivatives can inhibit human immunodeficiency virus type 1 (HIV-1) entry or maturation depending on side chain modifications. While BA derivatives with antimaturation activity have attracted considerable interest, the anti-HIV-1 profile and molecular mechanism of BA derivatives with anti-HIV-1 entry activity (termed BA entry inhibitors) have not been well defined. In this study, we have found that two BA entry inhibitors, IC9564 and A43D, exhibited a broad spectrum of anti-HIV-1 activity. Both compounds inhibited multiple strains of HIV-1 from clades A, B, and C at submicromolar concentrations. Clade C viruses were more sensitive to the compounds than clade A and B viruses. Interestingly, IC9564 at subinhibitory concentrations could alter the antifusion activities of other entry inhibitors. IC9564 was especially potent in increasing the sensitivity of HIV-1 YU2 Env-mediated membrane fusion to the CCR5 inhibitor TAK-779. Results from this study suggest that the V3 loop of gp120 is a critical determinant for the anti-HIV-1 activity of IC9564. IC9564 escape viruses contained mutations near the tip of the V3 loop. Moreover, IC9564 could compete with the binding of V3 monoclonal antibodies 447-52D and 39F. IC9564 also competed with the binding of gp120/CD4 complexes to chemokine receptors. In summary, these results suggest that BA entry inhibitors can potently inhibit a broad spectrum of primary HIV-1 isolates by targeting the V3 loop of gp120.     

env chimeric virus technology for evaluating human immunodeficiency virus susceptibility to entry inhibitors

We describe the development of chimeric virus technology (CVT) for human immunodeficiency virus (HIV) type 1 (HIV-1) env genes gp120, gp41, and gp160 for evaluation of the susceptibilities of HIV to entry inhibitors. This env CVT allows the recombination of env sequences derived from different strains into a proviral wild-type HIV-1 clone (clone NL4.3) from which the corresponding env gene has been deleted. An HIV-1 strain (strain NL4.3) resistant to the fusion inhibitor T20 (strain NL4.3/T20) was selected in vitro in the presence of T20. AMD3100-resistant strain NL3.4 (strain NL4.3/AMD3100) was previously selected by De Vreese et al. (K. De Vreese et al., J. Virol. 70:689-696, 1996). NL4.3/AMD3100 contains several mutations in its gp120 gene (De Vreese et al., J. Virol. 70:689-696, 1996), whereas NL4.3/T20 has mutations in both gp120 and gp41. Phenotypic analysis revealed that NL4.3/AMD3100 lost its susceptibility to dextran sulfate, AMD3100, AMD2763, T134, and T140 but not its susceptibility to T20, whereas NL4.3/T20 lost its susceptibility only to the inhibitory effect of T20. The recombination of gp120 of NL4.3/AMD3100 and gp41 of NL4.3/T20 or recombination of the gp160 genes of both strains into a wild-type background reproduced the phenotypic (cross-)resistance profiles of the corresponding strains selected in vitro. These data imply that mutations in gp120 alone are sufficient to reproduce the resistance profile of NL4.3/AMD3100. The same can be said for gp41 in relation to NL4.3/T20. In conclusion, we demonstrate the use of env CVT as a research tool in the delineation of the region important for the phenotypic (cross-)resistance of HIV strains to entry inhibitors. In addition, we obtained a proof of principle that env CVT can become a helpful diagnostic tool in assessments of the phenotypic resistance of clinical HIV isolates to HIV entry inhibitors.     

Conformational changes induced in the human immunodeficiency virus envelope glycoprotein by soluble CD4 binding

The human immunodeficiency virus (HIV) binds to the surface of T lymphocytes and other cells of the immune system via a high affinity interaction between CD4 and the HIV outer envelope glycoprotein, gp120. By analogy with certain other enveloped viruses, receptor binding by HIV may be followed by exposure of the hydrophobic NH2 terminus of its transmembrane glycoprotein, gp41, and fusion of the virus and cell membranes. A similar sequence of events is thought to take place between HIV-infected and uninfected CD4+ cells, resulting in their fusion to form syncytia. In this study, we have used a soluble, recombinant form of CD4 (sCD4) to model events taking place after receptor binding by the HIV envelope glycoproteins. We demonstrate that the complexing of sCD4 with gp120 induces conformational changes within envelope glycoprotein oligomers. This was measured on HIV-1-infected cells by the increased binding of antibodies to the gp120/V3 loops, and on the surface of virions by increased cleavage of this loop by an exogenous proteinase. At 37 degrees C, these conformational changes are coordinate with the dissociation of gp120/sCD4 complexes from gp41, and the increased exposure of gp41 epitopes. At 4 degrees C, gp120 dissociation from the cell surface does not occur, but increased exposure of both gp120/V3 and gp41 epitopes is detected. We propose that these events occurring after CD4 binding are integral components of the membrane fusion reaction between HIV or HIV-infected cells and CD4+ cells.     

Characterization of siamycin I, a human immunodeficiency virus fusion inhibitor.

The human immunodeficiency virus (HIV) fusion inhibitor siamycin I, a 21-residue tricyclic peptide, was identified from a Streptomyces culture by using a cell fusion assay involving cocultivation of HeLa-CD4+ cells and monkey kidney (BSC-1) cells expressing the HIV envelope gp160. Siamycin I is effective against acute HIV type 1 (HIV-1) and HIV-2 infections, with 50% effective doses ranging from 0.05 to 5.7 microM, and the concentration resulting in a 50% decrease in cell viability in the absence of viral infection is 150 microM in CEM-SS cells. Siamycin I inhibits fusion between C8166 cells and CEM-SS cells chronically infected with HIV (50% effective dose of 0.08 microM) but has no effect on Sendai virus-induced fusion or murine myoblast fusion. Siamycin I does not inhibit gp120 binding to CD4 in either gp120- or CD4-based capture enzyme-linked immunosorbent assays. Inhibition of HIV-induced fusion by this compound is reversible, suggesting that siamycin I binds noncovalently. An HIV-1 resistant variant was selected by in vitro passage of virus in the presence of increasing concentrations of siamycin I. Drug susceptibility studies on a chimeric virus containing the envelope gene from the siamycin I-resistant variant indicate that resistance maps to the gp160 gene. Envelope-deficient HIV complemented with gp160 from siamycin I-resistant HIV also displayed a resistant phenotype upon infection of HeLa-CD4-LTR-beta-gal cells. A comparison of the DNA sequences of the envelope genes from the resistant and parent viruses revealed a total of six amino acid changes. Together these results indicate that siamycin I interacts with the HIV envelope protein.     

Mutations in both env and gag genes are required for HIV-1 resistance to the polysulfonic dendrimer SPL2923, as corroborated by chimeric virus technology

A drug-resistant NL4.3/SPL2923 strain has previously been generated by in vitro selection of HIV-1(NL4.3) in the presence of the polysulfonic dendrimer SPL2923 and mutations were reported in its gp120 gene (Witvrouw et al., 2000). Here, we further analysed the (cross) resistance profile of NL4.3/SPL2923. NL4.3/SPL2923 was found to contain additional mutations in gp41 and showed reduced susceptibility to SPL2923, dextran sulfate (DS) and enfuvirtide. To delineate to what extent the mutations in each env gene were accountable for the phenotypic (cross) resistance of NL4.3/SPL2923, the gp120-, gp41- and gp160-sequences derived from this strain were placed into a wild-type background using env chimeric virus technology (CVT). The cross resistance of NL4.3/SPL2923 towards DS was fully reproduced following gp160-recombination, while it was only partially reproduced following gp120- or gp41-recombination. The mutations in gp41 of NL4.3/SPL2923 were sufficient to reproduce the cross resistance to enfuvirtide. Unexpectedly, the reduced sensitivity towards SPL2923 was not fully reproduced after gp160-recombination. The search for mutations in NL4.3/SPL2923 in viral genes other than env revealed several mutations in the gene encoding the HIV p17 matrix protein (MA) and one mutation in the gene encoding the p24 capsid protein (CA). In order to analyse the impact of the gag mutations alone and in combination with the mutations in env on the phenotypic resistance towards SPL2923, we developed a novel p17- and p17/gp160-CVT. Phenotypic analysis of the NL4.3/SPL2923 p17- and p17/gp160-recombined strains indicated that the mutations in both env and gag have to be present to fully reproduce the resistance of NL4.3/SPL2923 towards SPL2923.     

Neutralizing antibodies to human immunodeficiency virus type-1 gp120 induce envelope glycoprotein subunit dissociation

The spectrum of the anti-human immunodeficiency virus (HIV) neutralizing immune response has been analyzed by the production and characterization of monoclonal antibodies (mAbs) to the viral envelope glycoproteins, gp41 and gp120. Little is known, however, about the neutralization mechanism of these antibodies. Here we show that the binding of a group of neutralizing mAbs that react with regions of the gp120 molecule associated with and including the V2 and V3 loops, the C4 domain and supporting structures, induce the dissociation of gp120 from gp41 on cells infected with the T cell line-adapted HIV-1 molecular clone Hx10. Similar to soluble receptor-induced dissociation of gp120 from gp41, the antibody-induced dissociation is dose- and time- dependent. By contrast, mAbs binding to discontinuous epitopes overlapping the CD4 binding site do not induce gp120 dissociation, implying that mAb induced conformational changes in gp120 are epitope specific, and that HIV neutralization probably involves several mechanisms.     

Novel anti-CD4 monoclonal antibodies separate human immunodeficiency virus infection and fusion of CD4+ cells from virus binding

Human immunodeficiency virus (HIV) binds to cells via an interaction between CD4 and the virus envelope glycoprotein, gp120. Previous studies have localized the high affinity binding site for gp120 to the first domain of CD4, and monoclonal antibodies (mAbs) reactive with this region compete with gp120 binding and thereby block virus infectivity and syncytium formation. Despite a detailed understanding of the binding of gp120 to CD4, little is known of subsequent events leading to membrane fusion and virus entry. We describe two new mAbs reactive with the third domain of CD4 that inhibit steps subsequent to virus binding critical for HIV infectivity and cell fusion. Binding of recombinant gp120 or virus to CD4 is not inhibited by these antibodies, whereas infection and syncytium formation by a number of HIV isolates are blocked. These findings demonstrate that in addition to virus binding, CD4 may have an active role in membrane fusion.     

Sequential immunizations with rgp120s from independent isolates of human immunodeficiency virus type 1 induce the preferential expansion of broadly crossreactive B cells

The gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is a dominant target against which the host's humoral immune response is directed. Unfortunately, gp120 proteins from different isolates of HIV are antigenically distinct, complicating the use of the envelope glycoprotein in vaccines designed to prevent acquired immunodeficiency syndrome. Using an enzyme-linked immunosorbent spot assay (ELISA), BALB/c mice immunized and boosted with recombinant purified gp120 were studied at the single cell level for their humoral immune response to HIV-1 envelope proteins. Approximately 90% of responding B cells produced antibodies reactive with the immunizing form of gp120 but not with gp120s from other strains of HIV. A novel sandwich ELISA was then used to analyze the frequency with which individual in vivo activated B cells produced antibodies that crossreacted with heterologous gp120s. Repeated immunizations with a single gp120 or with a mixture of different gp120s resulted in the activation of primarily mono-specific (noncrossreactive) B cells. In contrast, the sequential immunization of mice with recombinant purified envelope proteins from different strains of HIV (IIIB, SF2, and Zr6) induced the selective expansion of B cells producing highly crossreactive antibodies.     

Conversion of an immunogenic human immunodeficiency virus (HIV) envelope synthetic peptide to a tolerogen in chimpanzees by the fusogenic domain of HIV gp41 envelope protein

The fusogenic (F) domain of human immunodeficiency virus (HIV) gp41 envelope (env) protein has sequence similarities to many virus and mediates the fusion of HIV-infected cells. During a survey of the immunogenicity of HIV env peptides in chimpanzees, we have observed that HIV peptide immunogenicity was dramatically altered by the NH2- terminal synthesis of the gp41 F domain to an otherwise immunogenic peptide. We compared two hybrid peptide types comprised of T helper (Th) and B cell epitopes of HIV gp120 env protein for their immunogenicity in chimpanzees. The Th-B epitope hybrid peptides contained the HIV gp120 Th cell determinant, T1 (amino acids [aa] 428- 440)-synthesized NH2 terminal to gp120 V3 loop peptides, which contain B cell epitopes that induce anti-HIV-neutralizing antibodies (SP10IIIB [aa 303-321] and SP10IIIB [A] [aa 303-327]). The F-Th-B peptide contained the HIV gp41 F domain of HIVIIIB gp41 (aa 519-530)- synthesized NH2 terminal to the Th-B peptide. Whereas Th-B peptides were potent immunogens for chimpanzee antibody and T cell-proliferative responses, the F-Th-B peptide induced lower anti-HIV gp120 T and B cell responses. Moreover, immunization of chimpanzees with F-Th-B peptide but not Th-B peptides induced a significant decrease in peripheral blood T lymphocytes (mean decrease during immunization, 52%; p < 0.02). Chimpanzees previously immunized with F-Th-B peptide did not respond well to immunization with Th-B peptide with T or B cell responses to HIV peptides, demonstrating that the F-Th-B peptide induced immune hyporesponsiveness to Th and B HIV gp120 env determinants. These observations raise the hypothesis that the HIV gp41 env F domain may be a biologically active immunoregulatory peptide in vivo, and by an as yet uncharacterized mechanism, promotes primate immune system hyporesponsiveness to otherwise immunogenic peptides.     

Construction of a binding site for human immunodeficiency virus type 1 gp120 in rat CD4

The human immunodeficiency virus (HIV-1) infects T lymphocytes via an interaction between the virus envelope glycoprotein gp120 and the CD4 antigen of T helper cells. Previous studies demonstrated that mutations in various regions of CD4 domain 1 lead to the loss of gp120 binding. In the present study the gp120 binding site was constructed in rat CD4 by replacing rat with human CD4 sequence. A series of mutants was constructed the best of which bound gp120 with an affinity only twofold less than that of human CD4. The data indicate that the gp120 binding site of human CD4 is constituted by residues 33-58 of domain 1.     

Structure and function of oligosaccharide chains of the envelope glycoprotein gp120 of human immunodeficiency virus type 1]

The surface of the human immunodeficiency virus (HIV-1), a causative agent for acquired immunodeficiency syndrome (AIDS), is covered with the major envelope glycoprotein gp120, of which the carbohydrate moiety accounted for 50% of the molecular mass. There is evidence that glycosylation of gp120 is prerequisite to the various stages of HIV infection. The oligosaccharide structures of gp120 have been determined using recombinant gp120 of HIV-1 (IIIB) produced in chinese hamster ovary cells and virus-derived gp120 isolated from H9 lymphocytes chronically infected with HIV-1 (IIIB). Three oligosaccharides have been suggested to be involved in the HIV-infection process. Occurrence of infection process which is independent of CD4 recognition and mediated by gp120 oligosaccharides, mannose-binding protein, and complement system has been suggested.     

The Phthalocyanine Prototype Derivative Alcian Blue Is the First Synthetic Agent with Selective Anti-Human Immunodeficiency Virus Activity Due to Its gp120 Glycan-Binding Potential

Alcian Blue (AB), a phthalocyanine derivative, is able to prevent infection by a wide spectrum of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strains in various cell types [T cells, (co)receptor-transfected cells, and peripheral blood mononuclear cells]. With the exception of herpes simplex virus, AB is inactive against a broad variety of other (DNA and RNA) viruses. Time-of-addition studies show that AB prevents HIV-1 infection at the virus entry stage, exactly at the same time as carbohydrate-binding agents do. AB also efficiently prevents fusion between persistently HIV-1-infected HUT-78 cells and uninfected (CD4⁺) lymphocytes, DC-SIGN-directed HIV-1 capture, and subsequent transmission to uninfected (CD4⁺) T lymphocytes. Prolonged passaging of HIV-1 at dose-escalating concentrations of AB resulted in the selection of mutant virus strains in which several N-glycans of the HIV-1 gp120 envelope were deleted and in which positively charged amino acid mutations in both gp120 and gp41 appeared. A mutant virus strain in which four N-glycans were deleted showed a 10-fold decrease in sensitivity to the inhibitory effect of AB. These data suggest that AB is likely endowed with carbohydrate-binding properties and can be considered an important lead compound in the development of novel synthetic nonpeptidic antiviral drugs targeting the glycans of the envelope of HIV.Targeting the entry process of human immunodeficiency virus (HIV), including drugs that bind to the receptor CD4, to a coreceptor, CCR5 or CXCR4, or to gp160 envelope is a valuable approach to prevent or suppress HIV infections. The first clinically used entry inhibitor, enfuvirtide (T20; Fuzeon) binds to the transmembrane gp41 of the envelope of HIV (21), thus preventing the required conformational changes of the envelope to successfully complete viral entry. Most recently, the CCR5 antagonist maraviroc was approved by the FDA for the treatment of HIV-infected individuals (16). Both enfuvirtide and maraviroc prove that HIV entry can be efficiently targeted by drugs that act at different stages in the entry process.The envelope of HIV consists of two subunits: the surface gp120 and the transmembrane gp41. Both units are highly glycosylated (23, 24), which is essential for the virus to escape immune surveillance (28). A broad variety of carbohydrate-binding agents (CBAs), such as the plant lectins Hippeastrum hybrid agglutinin (HHA), Galanthus nivalis agglutinin (GNA), and Urtica dioica agglutinin (UDA) or the prokaryotic cyanovirin-N (CV-N) and actinohivin, bind to the glycans that are present on the envelope of HIV and inhibit the viral entry process (4, 5). The majority of the natural CBAs are proteins (i.e., lectins), which may have some major drawbacks: (i) it is technically not easy and it is relatively costly to produce and purify these proteins on a large scale, (ii) they have poor, if any, oral bioavailability, and (iii) they can trigger an immune response that compromises their eventual antiviral efficacy (1). Therefore, (semi)synthetic low-molecular-weight compounds that are also able to bind glycans and prevent virus infection would be a valuable alternative.Recently, pradimicin A (PRM-A), an antifungal nonpeptidic antibiotic (26), was described to possess lectin-like properties and bind to the glycans of HIV gp120 (32) and proved able to efficiently prevent HIV infection (31). HIV selected under escalating PRM-A concentrations can escape this drug pressure by deleting multiple N-glycosylation sites in gp120 (10). This was earlier also observed to occur in HIV-1 strains selected under pressure of peptidic CBAs, such as HHA, GNA, UDA, and cyanovirin-N (5, 6). Thus, the CBAs not only prevent virus infection by binding to glycans on the envelope of HIV, but also they can force the virus to delete its envelope N-glycans in order to escape drug pressure, resulting in the exposure of previously hidden immunogenic epitopes. This phenomenon is interesting given the fact that the glycans on the HIV gp120 envelope play a very important role in shielding the virus from the immune system and in limiting the neutralizing antibody response to HIV (39).Here, we report on a synthetic compound, the phthalocyanine Alcian Blue (AB), that is endowed with anti-HIV activity due to its lectin-like properties. It prevents entry of HIV into its target cells and selects for mutant virus strains that have several deletions in N-glycosylation sites in gp120.     

Anti-HIV-1 activity of enfuvirtide (T-20) by inhibition of bystander cell death

Infection by human immunodeficiency virus type 1 (HIV-1) has been associated with increased cell death of both infected and bystander cells. The envelope glycoprotein complex appears to play an active role in HIV-induced death of bystander cells. We quantified cell-to-cell fusion, single cell death and membrane lipid mixing in cocultures of effector, HIV-1 envelope-expressing cells with peripheral blood mononuclear cells or purified CD4 T lymphocytes from HIV-negative donors, in the presence or the absence of the fusion inhibitor enfuvirtide (T-20, pentafuside, Fuzeon). T-20, which blocks gp41-dependent virus-cell fusion, showed a complete and dose-dependent inhibition of syncytium formation in cocultures of envelope-expressing cells with uninfected cells. Similarly, T-20 totally abrogated death of single bystander CD4 T cells with an IC50 of 0.04 microg/ml. Membrane lipid mixing, as a measure of interaction between envelope-expressing cells and CD4 cells, was also dose-dependently inhibited by T-20. Moreover, effector cells chronically infected with a T-20-resistant virus recovered the ability to induce bystander cell death in the presence of the drug, supporting the role of gp41 in single cell death. In conclusion, T-20 is able to protect CD4 T cells from envelope presentation with a dual effect: inhibition of virus replication and blockade of HIV-1 envelope-induced cell death of bystander CD4 T cells. Protection of cells prior to infection from HIV envelope-dependent bystander effect could lead to a better immune restoration of HIV-1-infected patients that are treated with T-20.     

Identification and characterization of UK-201844, a novel inhibitor that interferes with human immunodeficiency virus type 1 gp160 processing

More than 10(6) compounds were evaluated in a human immunodeficiency virus type 1 (HIV-1) high-throughput antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (UK-201844). UK-201844 exhibited antiviral activity against HIV-1 NL4-3 in MT-2 and PM1 cells, with 50% effective concentrations of 1.3 and 2.7 microM, respectively, but did not exhibit measurable antiviral activity against the closely related HIV-1 IIIB laboratory strain. UK-201844 specifically inhibited the production of infectious virions packaged with an HIV-1 envelope (Env), but not HIV virions packaged with a heterologous Env (i.e., the vesicular stomatitis virus glycoprotein), suggesting that the compound targets HIV-1 Env late in infection. Subsequent antiviral assays using HIV-1 NL4-3/IIIB chimeric viruses showed that HIV-1 Env sequences were critical determinants of UK-201844 susceptibility. Consistent with this, in vitro resistant-virus studies revealed that amino acid substitutions in HIV-1 Env are sufficient to confer resistance to UK-201844. Western analysis of HIV Env proteins expressed in transfected cells or in isolated virions showed that UK-201844 inhibited HIV-1 gp160 processing, resulting in the production of virions with nonfunctional Env glycoproteins. Our results demonstrate that UK-201844 represents the prototype for a unique HIV-1 inhibitor class that directly or indirectly interferes with HIV-1 gp160 processing.     

Cyanovirin-N, a potent human immunodeficiency virus-inactivating protein, blocks both CD4-dependent and CD4-independent binding of soluble gp120 (sgp120) to target cells, inhibits sCD4-induced binding of sgp120 to cell-associated CXCR4, and dissociates bound sgp120 from target cells

Cyanovirin-N (CV-N), an 11-kDa protein originally isolated from the cyanobacterium Nostoc ellipsosporum, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, simian immunodeficiency virus, and feline immunodeficiency virus. It has been well established that the HIV surface envelope glycoprotein gp120 is a molecular target of CV-N. We recently reported that CV-N impaired the binding of virion-associated gp120 to cell-associated CD4 and that CV-N preferentially inhibited binding of the glycosylation-dependent neutralizing monoclonal antibody 2G12 to gp120. However, CV-N did not interfere with the interactions of soluble CD4 (sCD4) with either soluble gp120 (sgp120) or virion-associated gp120. In the present study, we have evaluated the effects of CV-N on the binding of sgp120 to cell-associated CD4 to clarify the experimental basis of the previous binding results, and we further address the detailed mechanism of action of CV-N. Here we present evidence that (i) CV-N impairs both CD4-dependent and CD4-independent binding of sgp120 to the target cells, (ii) CV-N blocks the sCD4-induced binding of sgp120 with cell-associated coreceptor CXCR4, and (iii) CV-N dissociates bound sgp120 from target cells. The results illustrate that the measured effects of CV-N on gp120-CD4 binding interactions depend upon the type of CD4 (soluble or cell associated), but not upon the type of gp120 (soluble or virion associated), employed in the experimental protocol. In addition, this study reinforces that CV-N acts uniquely to prevent essential interactions between the envelope glycoprotein and target cell receptors and further supports the potential broad utility of CV-N as a microbicide to prevent the transmission of HIV and AIDS.     

ADS-J1 Inhibits HIV-1 Entry by Interacting with gp120 and Does Not Block Fusion-Active gp41 Core Formation

We had shown that virus resistance to ADS-J1 was associated with amino acid changes in the envelope glycoprotein, mostly located in the gp120 coding region. Time-of-addition and endocytic virus transfer assays clearly demonstrated that ADS-J1 behaved as a gp120 inhibitor. ADS-J1-resistant virus was cross-resistant to the polyanion dextran sulfate, and recombination of gp120 recovered only the ADS-J1-resistant phenotype. In summary, ADS-J1 blocks an early step of virus entry that appears to be driven by gp120 alone.The essential steps of HIV-1 entry in the host cell offer several potential targets for the development of novel antiviral agents (19, 24, 33, 42). Agents that disrupt gp41-mediated membrane fusion, collectively called fusion inhibitors, were the first entry inhibitors to be approved for the treatment of HIV infection. Enfuvirtide (T20, Fuzeon) is a 36-amino-acid synthetic peptide with a sequence identical to a part of the C-terminal heptad repeat 2 (HR2) region of gp41 that binds to the N-terminal heptad repeat 1 (HR1) in an antiparallel manner, forming a coiled-coil structure during the prefusion step. Mutations in the highly conserved amino acid motif 36 to 45 in the HR1 domain confer resistance to T20 (35), providing strong evidence that HR1 is the site of interaction of T20. However, mutations in other regions of HIV-1 envelope (Env) have been also associated with T20 resistance (26, 27).Several low-molecular-weight (SMW) compounds have been identified as blockers of the initial steps of virus entry, including CCR5 coreceptor (33, 42). However, the identification of SMW compounds targeting gp41 has been elusive. A polyanionic compound, ADS-J1, was previously identified in silico as a potential candidate and shown to bind to gp41 peptides and interfere with the formation of the gp41 coiled-coil domain in an in vitro enzyme-linked immunosorbent assay (ELISA) model of HR1-HR2 interaction (16, 30, 31). Conversely, we had shown that ADS-J1 blocked the binding of HIV particles to lymphoid MT-4 cells and inhibited HIV replication at a time/site of interaction similar to those of the polyanion dextran sulfate (DS), a well-described, nonspecific inhibitor of virus entry (3). Moreover, at least four HIV-1 strains resistant to ADS-J1 were generated. The resistance to ADS-J1 was associated with gp120 based on the fact that the majority of the mutations were located in the gp120 coding sequence, mainly in the V3 loop region. Although three of the resistant strains contained mutations in gp41, one of them, HIV-1 ARA45C, did not (3). In addition, molecular modeling suggested that the gp120 V3 loop was the preferential binding site for ADS-J1 onto HIV-1, and mutations induced by the inhibitor significantly changed the stereoelectronic properties of the gp120 surface, justifying a marked drop in the affinity of ADS-J1 toward an ADS-J1-resistant HIV-1 strain (36). At that time, we considered conclusive the evidence of the mode of action of ADS-J1.More recently, Wang et al. (43) suggested that ADS-J1 could bind directly to a trimeric peptide containing the gp41 pocket region (IQN17) in a surface plasmon resonance (SPR) assay and indicated that ADS-J1 can be used as a lead compound for the design of novel HIV-1 fusion inhibitors (44). Therefore, we thought it relevant to provide further evidence of the mode of action of ADS-J1.     

Phosphorothioate oligonucleotides inhibit human immunodeficiency virus type 1 fusion by blocking gp41 core formation

Several studies have shown that phosphorothioate oligodeoxynucleotides (PS-ONs) have a sequence-independent antiviral activity against human immunodeficiency virus type 1 (HIV-1). It has also been suggested that PS-ONs inhibit HIV-1 by acting as attachment inhibitors that bind to the V3 loop of gp120 and prevent the gp120-CD4 interaction. Here we show that PS-ONs (and their fully 2'-O-methylated derivatives) are potent inhibitors of HIV-1-mediated membrane fusion and HIV-1 replication in a size-dependent, phosphorothioation-dependent manner. PS-ONs interact with a peptide derived from the N-terminal heptad repeat region of gp41, and the HIV-1 fusion-inhibitory activity of PS-ONs is closely correlated with their ability to block gp41 six-helix bundle formation, a critical step during the process of HIV-1 fusion with the target cell. These results suggest that the increased hydrophobicity of PS-ONs may contribute to their inhibitory activity against HIV-1 fusion and entry, because longer PS-ONs (>or=30 bases) which have a greater hydrophobicity are more potent in blocking the hydrophobic interactions involved in the gp41 six-helix bundle formation and inhibiting the HIV-1-mediated cell-cell fusion than shorter PS-ONs (<30 bases). This novel antiviral mechanism of action of long PS-ONs has implications for therapy against infection by HIV-1 and other enveloped viruses with type I fusion proteins.     

Processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1 in the presence of trimming glucosidase inhibitor deoxynojirimycin

The processing and secretion of the envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) were studied in chronically infected cells treated with the trimming glucosidase inhibitor deoxynojirimycin (DNM). In Molt3 cells infected with human T-lymphotropic virus type III (HTLV-IIIB), DNM inhibited the intracellular proteolytic processing of gp160 to gp120 and gp41. A clone of the HUT78 cell line called 6D5, when chronically infected with the HIV-1 isolate HTLV-III451 was shown to release both gp160 and gp120 into the culture medium. The secretion of envelope glycoproteins from these infected cells was not inhibited by DNM treatment. The secreted proteins had higher molecular weights than gp160 and gp120 from cultures not treated with DNM, presumably due to the presence of unprocessed carbohydrate residues on the polypeptide chain. These secreted glycoproteins from DNM-treated cells exhibited specific interaction with the CD4 molecule on the surface of target cells. However, the syncytium formation induced by HIV-1-infected cells on CD4+ cells was significantly inhibited in the presence of the glucosidase inhibitor. The minimal cytotoxicity of the DNM coupled with its strong inhibitory effect on the cell-to-cell spread of the virus suggest that it may be potentially useful in antiviral drug therapy of HIV-1 infection.